Implementación de dos métodos de recuento en placa para la detección de colifagos somáticos, aportes a las metodologías estándar

Abstract

Two plate count methods, double layer and single layer of agar for quantification of somatic coliphages in water, were implemented using standard methodologies. Several variables were tested and provided valuable information that was not included in standard methodologies. The most important findings are described, such as the effect of using an excessively concentrated culture of E. coliand production of a log phase culture in only 4 hours of incubation, adjusting the concentration to an optical density of 0.3 at 600 nm (3.1 x 10 8 CFU / mL), or to McFarland 1 (3.0 x 108 CFU / mL). It was determined that coliphages controls must be stored at -70 °C to reduce its degradation. Quantities of reaction mixture exceeding 20 mL per Petri dish must be avoided to prevent interfere with bubbles during the counting of plate forming units (PFU). It was demonstrated that coliphages isolated from water samples can be stored for 48 hours at 4 °C without any degradation, and PFU are not observed in samples with high concentrations of coliphages, because a confluent lysis of the bacte-rial layer. There was no significant difference in the recovery of coliphages using doble layer or single layer methods, but such methods should be evaluated by means of controls, before applying them directly in the analysis of water samples.

Evaluación temporal y espacial en la calidad microbiológica del agua superficial: caso en un sistema de abastecimiento de agua para consumo humano en Costa Rica

“…La cuantificación de colifagos somáticos se realizó utilizando el método de capa simple de agar descrito por Solano et al, (2012), el cual consiste en mezclar 100 mL de ATS 1.2 %, 5 mL de cloruro de magnesio y 10 mL de cultivo de E. coli ATCC 13906 en fase logarítmica, con una densidad óptica de 0.300 ± 0.050 y 100 mL de la muestra de agua previamente filtrada por filtros de 47 mm de acetato de celulosa de 0.2 µm de poro (Sartorius, Alemania), previamente tratados con extracto de carne pH 7.2 (Oxoid, Estados Unidos). La mezcla se homogenizó y se dispensó en platos Petri, los cuales se incubaron a 35.0 ±0.5 ºC durante veinticuatro horas.…”

A Somatic Coliphage Threshold Approach To Improve the Management of Activated Sludge Wastewater Treatment Plant Effluents in Resource-Limited Regions

“…Thus, the theoretical process limit of enteric virus detection for any given assay was estimated to range from 183 virus copies/100 mL to 18,300 copies/100 mL. that was pretreated with 3% beef extract pH 7.2 (33,44). Somatic coliphage concentrations were identified in WWTP effluent samples using single-layer plaque assay (undiluted sample) and in WWTP influent samples using double-layer plaque assay (1:10,000 serial-dilution of sample).…”